Some of the major advances in the area of carbohydrate chemistry are the development of methodologies to chemically synthesize complex oligosaccharides of defined sequences. The combination of the chemical and enzymatic syntheses of these materials affords greater possibilities to obtain oligosaccharides that cannot be made by means of chemical synthesis alone. Since there are commercially available monosaccharides, it is now possible to begin the synthesis of complex oligosaccharides with a starting sugar residue containing an appropriate tether and add many more complex sugars units as desired. Once the synthesis is completed, the oligosaccharide can be conveniently coupled via tethers to proteins. The methodology for synthesizing carbohydrate-tether conjugates has been described in U.S. Pat. No. 4,137,401. The general methodology for coupling ligands to proteins has been described by Inman et al., Immunochemistry 10, 165-174 (1973) and Stowell et al., Advances in Carbohydrate Chemistry and Biochemistry 37: 225-281, (1980).
Interleukin-2 (IL-2) is a protein secreted by lymphocytes which belongs to the class of immune modulating substances called lymphokines. IL-2 has been shown to modulate a number of immunological activities of lymphoid cells including cytotoxic T-cell activity, activation of natural killer (NK) cells, activation of B-cells, and generation of lymphokine activated killer (LAK) cells (cells that kill tumor cells but not normal cells). (Grimm et al., J Exp. Med., 155: 1823-1841 (1982); Mazumder et al., J. Exp. Med., 159: 495-507 (1984), Grimm et al., Lymphokines, 9:279-311 (1984)). Recently, treatment of cancer patients by administration of recombinant IL-2 (rIL-2) and autologous LAK cells has demonstrated the potential use of rIL-2 as an immunotherapeutic agent (Rosenberg et al., N. Eng. J. Med., 313: 1485-1492 (1985)). Rosenberg points out that in many patients, the administration of therapy was limited by the toxicity of rIL-2, and greater antitumor effects might occur if larger doses of rIL-2 and LAK cells could be administered. Rosenberg further teaches that rIL-2 can cause some of the side effects by possibly stimulating helper T cells to secrete other lymphokines, that may be toxic (Rosenberg et al., N. Eng. J. Med., 313:1485-1492 (1985)), Rosenberg et al., N. Eng. J. Med., 316:889-897 (1987).
Glycoproteins may be defined as "conjugated proteins containing as prosthetic group(s) one or more heterosaccharide(s), usually branched, with a relatively low number of sugar residues, lacking a serially repeating unit and bound covalently to the polypeptide chain." (Biochemistry and Molecular Biology, 1989, ed G.D. Fasman, CRC Press, Inc., 2000 Corporate Blvd., Boca Raton, Florida, 33431).
There are three classes of linkages between the carbohydrate and an amino acid of the protein portion in nature. One type has a N-glycosyl link to the amide nitrogen of asparnagine. A second type has a glycosidic bond between N-acetylgalactosamine and a serine or threonine hydroxyl, and a third has an attachment of the carbohydrate to the protein via the hydroxyl of hydroxylysine. (Carbohydrate Biochemistry and Metabolism, 1984, K. L. Roehrig, The AVI Publishing Company, Inc., Westport, Conn.).
Carbohydrates attached to proteins have been found to be essential in recognition or binding and to protect proteins against changes in temperature and pH (Schwartz et al., Adv. Carbohydr. Chem. Biochem., 40, 287-379 (1982)).
Natural IL-2 is a glycoprotein, with an estimated molecular weight of about 16,500 and contains carbohydrate O-linked to the threonine, the third amino acid from the amino terminal end (Conradt et al., Eur. J. Biochem., 153(2), 255-261 (1985). The gene responsible for the synthesis of human IL-2 has been cloned and sequenced (Taniguchi et al., Nature, 302: 305-310 (1983); Deves et al., Nucleic Acids Res., 11: 4307-4323 (1983)). The large quantities of IL-2 that are required for various clinical trials are currently produced as a result of cloning the gene for IL-2 and expressing it in E. coli (Ju et al., J. Biol. Chem., 262: 5723-5731 (1987). Even though the bacterially produced recombinant material lacks carbohydrates that are present on the natural material, it is functionally active. However, some of the physical properties of the bacterially produced material (rIL-2) are different from the native IL-2. The rIL-2 is produced as insoluble refractile bodies within the bacteria and therefore denaturants are required during its purification. In the absense of a detergent, purified rIL-2 has very limited solubility at neutral pH (Katre et al., Proc. Natl. Acad. Sci. USA, 84, 1487-1491 (1987). Due to rapid clearance, it also has a short circulatory serum half-life when administered to animals.
To delay the rate of in vivo clearance, EP 154,316 discloses chemically modified lymphokines, including rIL-2, containing polyethylene glycol bonded directly to at least one primary amino group of a lymphokine.
To overcome the limited solubility at neutral pH and the short circulatory half-life of rIL-2 purified from E. coli, Katre et al. and WO87/00056 describe the modification of the rIL-2 by conjugating it with monomethoxy polyethylene glycol. The modified rIL-2 had enhanced solubility decreased plasma clearance and increased antitumor potency in a particular animal tumor model.
In this invention, various mono- and oligosaccharide-tether-conjugates have been attached by chemical means via an amide bond to the amino group of one or more of the 11 lysines in rIL-2, a biologically active protein. For the purposes of the disclosures of this invention, the process of attaching a saccharide(s) to at least one lysine in the rIL-2 molecule is considered glycolysation and the product is termed lysine-glycosylated rIL-2.
The glycoslated rIL-2 preparations of this invention were more readily soluble in water than unglycosylated IL-2 while retaining their biological activity. One biologically active glycosylated rIL-2 preparation was tested and found to be thermally more stable than rIL-2 when heated up to 90.degree. C. Surprisingly, several glycosylated rIL-2 preparations which resulted from the method of this invention lost most of their T lymphocyte activating ability, while retaining most or all their biological activity, i.e., the ability to enhance NK cell and LAK cell activities.